Sulfotransferase assay

ABSTRACT

A method of detecting sulfotransferase activity including conducting a first reaction comprising, measuring free phosphate produced by the first reaction, and comparing the measured free phosphate to a free phosphate standard curve or equation or to a free phosphate level obtained in a separate reaction. The first reaction includes combining the sulfotransferase, Golgi-resident PAP-phosphatase (gPAPP), a 5′-nucleotidase, a substrate of the sulfotransferase, and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) under conditions to produce 3′-phosphoadenosine-5′-phosphate (PAP).

PRIORITY CLAIM

This application claims priority to U.S. patent application Ser. No.13/113,637, filed May 23, 2011, the disclosure of which is herebyincorporated by reference in the entirety.

BACKGROUND

Sulfation is a common modification that affects the biological activityof a wide variety of substrates. Sulfation reactions are catalyzed bysulfotransferases. Sulfotransferases are a large group of enzymes thattransfer a sulfate from a donor substrate to an acceptor. Manysulfotransferases exist in nature, but in humans, the sulfotransferasesare of two types. Cystosolic sulfotransferases (SULTs) are mainlyinvolved in modifying small molecules such as steroids,neurotransmitters, and xenobiotics, and in drug detoxification.Golgi-resident sulfotransferases are involved in modifying glycans andproteins on cell membranes and within the extracellular matrix. Sulfatedglycans such as glycosaminoglycans and numerous O- and N-glycans playroles in maintaining biochemical and biophysical properties. Sulfatedproteins, such as leukocyte adhesion molecule PSGL-1, play roles inprotein-protein and cellular interactions.

Because of the important roles of sulfated molecules in variousbiological events, sulfotransferases may be ideal targets for drugintervention. Several assays for detecting sulfotransferase activityexist, but they have significant drawbacks. Some sulfotransferase assaysuses a radioisotope, ³⁵S. Such assays require separation steps such asHPLC, TLC, filter blinding, or electrophoresis to separate substratesfrom products. As such, in addition to the difficulties associated withthe use of radioisotopes, the need for separation makes such assays verytime consuming. Other methods which can be used include massspectrometry, fluorescent detection and colorimetric detection. Onefluorescent method uses 4-methylumbellifery sulfate as the ultimatesulfate donor and measures the fluorescence of 4-methylumbelliferone.Similarly, one colorimetric detection method uses p-nitrophenyl sulfateas the ultimate sulfate donor and measures the color intensity of thegenerated p-nitrophenol. Each of these methods is either time consumingor lacks efficiency, limiting their usefulness. An assay which can beperformed easily and provides results rapidly would be ideal,particularly for screening potential drugs for their effect onsulfotransferases.

SUMMARY

Embodiments of the invention include assays, methods and kits fordetecting and quantifying sulfotransferase activity. In someembodiments, the method includes detecting sulfotransferase activityincluding conducting a first reaction by combining a sulfotransferase,Golgi-resident PAP-phosphatase (gPAPP), a 5′-nucleotidase, a substrateof the sulfotransferase, and 3′-phosphoadenosine-5′-phosphosulfate(PAPS) under conditions to produce 3′-phosphoadenosine-5′-phosphate(PAP), measuring free phosphate, and comparing the measured freephosphate to a free phosphate standard curve or equation or to a freephosphate level obtained in a separate reaction. The method may furtherinclude calculating sulfotransferase activity using the measured amountof free phosphate.

In some embodiments, the separate reaction includes a background controlreaction wherein the (gPAPP), the 5′-nucleotidase, the substrate of thesulfotransferase, and the (PAPS) were combined without thesulfotransferase. The method may further include reducing the measuredphosphate of the reaction by the measured phosphate of the backgroundcontrol reaction to calculate the amount of phosphate correlated to thesulfotransferase reaction. The method may include calculatingsulfotransferase activity using the calculated the amount of phosphatecorrelated to the sulfotransferase reaction.

In some embodiments, the separate reaction comprises a reaction whereinthe sulfotransferase, the gPAPP, the substrate of the sulfotransferase,and the PAPS were combined and wherein one or more conditions of theseparate reaction were different from those of the reaction.

In some embodiments, measuring the free phosphate includes measuringoptical density. In some embodiments, measuring free phosphate includesapplying a colorimetric free phosphate detection assay to the firstreaction. For example, measuring free phosphate may include adding afirst reagent to the reaction comprising ammonium molybdate and thenadding a second reagent to the reaction comprising malachite greenoxalate.

FIGURES

FIG. 1 is a representative sulfotransferase reaction;

FIG. 2 is a sulfotransferase reaction including release of freephosphate according to embodiments of the invention;

FIG. 3 is another sulfotransferase reaction including release of freephosphate according to embodiments of the invention;

FIG. 4 is a graph of gPAPP activity versus MgCl₂ concentration;

FIG. 5 is a graph of the ratio of free phosphate released by gPAPPand/or CD73 (Pi) to the free phosphate released by TNAP (Pi_(TNAP)) from3′-AMP, 5′-AMP and PAP;

FIG. 6 is a graph of phosphate released from PAPS and PAP versus gPAPPconcentration;

FIG. 7 is a graph of phosphate released from PAP by gPAPP in thepresence or absence of PAPS versus PAP concentration;

FIG. 8 is a graph of the rate of phosphate release versus SULT1A1concentration under various conditions;

FIG. 9 is a graph of the activity of SULT1A1 versus concentration ofpNP;

FIG. 10A is a graph of activity of CHST3 versus concentration of CS;

FIG. 10B is a graph of activity of CHST3 versus concentration of PAPS;and

FIG. 11 is a graph of CHST3 activity versus CHST3 concentration.

DETAILED DESCRIPTION

The following detailed description is exemplary in nature and is notintended to limit the scope, applicability, or configuration of theinvention in any way. Rather, the following description providespractical illustrations for implementing exemplary embodiments of thepresent invention. Examples of constructions, materials, dimensions, andmanufacturing processes are provided for selected elements, and allother elements employ that which is known to those of skill in the fieldof the invention. Those skilled in the art will recognize that many ofthe examples provided have suitable alternatives that can be utilized.

Embodiments of the invention provide methods and assays for quickly andeasily detecting and quantifying the activity of sulfotransferaseenzymes. Embodiments of the invention are useful for high throughputtesting of drugs to evaluate their effect upon sulfotransferases.

Embodiments of the invention utilize the nearly universal use of PAPS asthe sulfate donor in sulfotransferase reactions, with ultimate releaseand detection of the phosphate in the PAPS using a colorimetric assay.

To better understand the embodiments of the invention, a genericsulfation reaction is shown in FIG. 1. The sustrate is shown as R. Aftersulfation by the sulfotransferase, the sulfated product is shown as S-R.In nearly all sulfation reactions, the sulfate donor is PAPS, and thissulfate donor is shown. After the transfer of the sulfate to thesubstrate S, it can be seen that the PAPS looses its sulfate and becomesPAP. Each sulfation reaction therefore results in the production of 1molecule of PAP. Embodiments of the invention rely upon this one to onenature of the relationship between PAP production and sulfation, and thenearly universal nature of PAPS as the sulfate donor, to provide amethod to detect and quantify sulfation as further described below. Itcan also be seen that the removal of the sulfate from PAPS results inthe exposure of the 3′ phosphate of PAP, which makes the 3′ phosphateavailable for release by a gPAPP.

FIG. 2 shows how embodiments of the invention can be used to assaysulfotransferase activity by utilizing the production of PAP from PAPS.The sulfotransferase reaction proceeds as shown in FIG. 1, with theproduction of PAP in direct correlation to sulfotransferase activity.The PAP then reacts with a PAP specific phosphatase, gPAPP, to releasethe 3′ phosphate from the PAP and generate 1 molecule of 5-adenosinemonophosphate (5′-AMP) and one molecule of free phosphate. The quantityof the free phosphate produced can then be detected by one of variousknown free phosphate detectors, and this amount directly correlates tothe activity of the sulfotransferase. In some embodiments, the freephosphate detector is a colorimetric assay, which is particularly usefulfor high throughput testing.

Embodiments of the invention may be used with any sulfotransferase whichuses PAPS as the sulfate donor. This includes nearly all knownsulfotransferases, including all human and all other mammaliansulfotransferases and some bacterial sulfotransferases. Indeed, allsulfotransferases, except for a small number present in bacteria, usePAPS as the sulfate donor. Examples of specific sulfotransferases whichcan be used in embodiments of the invention include carbohydratesulfotransferases (such as CHST1, CHST2, CHST3, CHST4, CHST5, CHST6,CHST7, CHST8, CHST9, CHST10, CHST11, CHST12, CHS13, CHS14 and CHS15),galactose-3-o-sulftotransferases (such as GAL3ST1, GAL3ST2, GAL3ST3, andGAL3ST4), heparin sulfate 2-O-sulfotransferases (such as HS2ST1),heparin sulfate 3-O-sulfotransferases (such as HS3ST1, HS3ST2, HS3ST3A1,HSEST3A2, HS3ST3B1, HS3ST3B2, HS3ST4, HS3ST5, and HS3ST6), heparinsulfate 6-O-sulfotransferases (such as HS6ST1, HS6ST2, and HS6ST3),N-deacetylase/N-sulfotransferases (such as NDST1, NDST2, NDST3, andNDST4), tyrosylprotein sulfotransferases (such as TPST1 and TPST2),uronyl-2-sulfotransferases (such as UST), estrone sulfotranferases,chondroitin 4-sulfotransferases, and others, such as SULT1A1, SULT1A2,SULT1A3, SULT1B1, SULT1C3, SULT1C4, SULT1DP, SULT1E1, SULT2A1, SULT2B1,SULT4A1 and SULT6B1. Embodiments of the invention can also be used withmicrobial sulfotransferases, such as Nod factor H, a sulfotransferase ofRhizobium melioti involved in establishing nitrogen-fixing symbiosisbetween rhizobia and leguminous plants, and StaL, a glycopeptideantibiotic sulfotransferase from Streptomyces toyocaensis.

The substrate used in embodiments of the invention is any substratewhich is acted upon by the sulfotransferase used in the assay. Thesubstrate may be a protein, carbohydrate, lipid or steroid, for example.Other examples of substrates include peptides, oligosaccharides, drugsand xenobiotics. For example, sulfation by cytosolic sulfotransferasescan be one step in the metabolism of certain drugs or xenobiotics.Therefore in some embodiments, a drug or xenobiotic may be used as asubstrate and the assay may be used to analyze the rate of sulfation ofthe drug or xenobiotic. In some embodiments, a drug or xenobiotic may beused as a substrate and an additional agent may be added to thereaction, such as a potential promoter or inhibitor of thesulfotransferase, to determine the effect of the additional agent uponthe metabolism of the drug or xenobiotic by the sulfotransferase.

Embodiments of the invention take advantage of the specificity of gPAPPas a coupling phosphatase. Both PAP and PAPS include phosphate moietiesthat could be removed by phosphatases. However, gPAPP is specificallyactive on PAP and not on PAPS. As such, all phosphate released by gPAPPis from PAP and not from PAPS. Because PAP is produced by thesulfotransferase reaction, the production of PAP directly correlates tothe activity of the sulfotransferase. The specificity of gPAPP thereforeallows phosphate to be released from PAP only, and this phosphate canthen be measured, such that the measured amount of phosphate directlycorrelates to the activity of the sulfotransferase.

The phosphatase gPAPP may be isolated from naturally occurring sourcesor may be produced recombinantly. The phosphatase gPAPP useful inembodiments of the invention also include any homologue proteins fromdifferent organisms and any mutational variations described herein. Onmethod of obtaining gPAPP which may be used in embodiments of theinvention is by using recombinant mouse gPAPP from E51 to K356 (Gene ID:242291) which can be expressed in CHO cells as an N-terminal His-taggedrecombinant protein and purified using nickel affinity resin andSuperdex-200 from GE Healthcare (Pittsburgh, Pa.). Further descriptionof how to make gPAPP which can be used in embodiments of the inventioncan be found in Frederick, J. P., et al. (2008) A role for alithium-inhibited Golgi nucleotidase in skeletal development andsulfation. Proc Natl Acad Sci USA. 105, 11605-11612, the relevantportions of which are hereby incorporated by reference. One method ofobtaining PAPS which can be used in embodiments of the invention it byusing recombinant S. cerevisiae ATP sulfurylase and P. chrysogenum APSkinase, then purigying the PAPS using a DEAE Sepharose fast-flow columnfrom GE Healthcare, for example. Further description of how to producePAPS which may be used in embodiments of the invention is provided inWu, Z. L., et al. A versatile polyacrylamide gel electrophoresis basedsulfotransferase assay. BMC biotechnology. 10,11, the relevant portionsof which are hereby incorporated by reference.

Some embodiments include the use of PAP, such as including PAP as acomponent of the assay. PAP which can be used in embodiments of theinvention is commercially available from Sigma Aldrich (St Louis, Mo.).After release of the phosphate from the PAP by gPAPP, the free phosphatemay be readily detected and/or measured by any means. Several methodsare known for measuring free phosphate, any of which may be used. Insome embodiments, the free phosphate may be detected and/or measuredusing a colorimetric assay. Examples of colorimetric assays formeasurement of free phosphate which may be used in embodiments of theinvention include the Malachite Green Phosphate Detection Kit availablefrom R & D Systems, (Minneapolis, Minn.), PiColorlock™ Assay reagentavailable from Innova Biosciences, Ltd. (Cambridge, U.K.), and PhosphateColorimetric Assay Kit available from BioVision (Mountain View, Calif.).In other embodiments, the free phosphate may be detected and/or measuredby fluorescence detection. For example, free phosphate may beselectively detected by a fluorescent sensor as described in U.S. Pat.No. 7,521,250, the disclosure of which is hereby incorporated byreference. In another example, free phosphate may be detected using arecombinant E. coli phosphate-binding protein labeled with thefluorophore MDCC known as Phosphate Sensor and available from Invitrogen(Carlsbad, Calif.).

The Malachite Green Phosphate Detection Kit is one method that may beused to detect free phosphate and is based on the malachitegreen-molybdate binding reaction, and the kit itself, or the componentsor variations thereof, may be used in embodiments of the invention. TheMalachite Green assay includes a first reagent, Malachite Green ReagentA, which includes ammonium molybdate and sulfuric acid, and a secondreagent, Malachite Green Reagent B, which includes malachite greenoxalate and polyvinyl alcohol. The Malachite Green assay furtherincludes a phosphate standard, KH₂PO₄. The phosphate standard may beused to create a standard curve of absorbance at 620 nm forinterpretation of sample assay results. The use of the assay includesincubating a sample with Malachite Green Reagent A for 10 minutes atroom temperature, then adding Malachite Green Reagent B and incubatingfor 20 minutes at room temperature. The absorbance may then be read at620 nm and compared to the phosphate standard curve to determine theamount of phosphate present in the sample.

The Malachite Green Phosphate Detection kit itself, or components orvariations thereof, may therefore be used to detect levels of freephosphate released from PAP, according to embodiments of the invention.In such embodiments, PAPS, substrate, sulfotransferase, assay buffer,and gPAPP are combined to produce a sample for testing. In someembodiments, the sample may further include an additional component ortest agent, such as a potential sulfotransferase inhibitor or promoter.At the completion of the reaction time, the resulting sample may becombined with Malachite Green Reagent A to stop the reaction, thenincubated 10 minutes, and then combined with Malachite Green Reagent Band incubated an additional 20 minutes as described above. Theabsorbance may then be read at 620 nm using a spectrometer, and thereading may be correlated to a phosphate standard curve and/or a control(including all reaction components except the sulfotransferase enzyme),to determine the amount of free phosphate released by gPAPP. This amountmay be compared to the initial quantity of PAPS and/or sulfotransferasepresent in the sample to determine the activity of the sulfotransferase.When a test agent is used, this amount may be compared to the amount ofphosphate produced in a reaction including all of the same componentsexcept the test agent, to determine the effect of the test agent uponthe sulfotransferase activity.

It should be noted that the PAP produced by the sulfatase reactionincludes two phosphate molecules, which are known as the 3′ and 5′phosphates. The phosphatase gPAPP is not only specific for PAP (ascompared to PAPS) but also is specific for the 3′ phosphate of PAP, suchthat gPAPP only releases the 3′ phosphate from PAP while the 5′phosphate is unaffected. In the reaction shown in FIG. 2, the 5′phosphate remains bound to the PAP and is not detected by the freephosphate detector. However, in some embodiments, the 5′ phosphate canalso be removed from PAP through the use of an additional specificphosphatase (a 5′-nucleotidase).

An example of a reaction in which two phosphates are released is shownin FIG. 3. The first portion of this reaction is the same as thereaction shown in FIG. 2, with PAPS converted to PAP during thesulfotransferase reaction, and gPAPP releasing one phosphate (the 3′phosphate) from each PAP produced. Removal of the 3′ phosphate from PAPproduces 5′-AMP. In this embodiment, the reaction also includes a5′-nucleotidase which removes the remaining phosphate from 5 ‘AMP toproduce an additional molecule of phosphate and an adenosine for eachsulfotransferase reaction.

Examples of 5′-nucleotidases which may be used in embodiments of theinvention include CD73, NT5DC1, NT5DC2, NT5DC3, cytosolic5′-nucleotidase IA (NT5C1A), cytosolic 5′-nucleotidase IB (NT5C1B),cytosolic 5′-nucleotidase II (NT5C2), cytosolic 5′-nucleotidase III(NT5C3), NT5C3L, NT5DC4. The 5 ‘-nucleotideases may be isolated fromnaturally occurring sources or may be produced recombinantly and manyare commercially available. The 5’-nucleotidases useful in embodimentsof the invention also include any homologue proteins from differentorganisms and any mutational variations of any of the phosphatasesdescribed herein.

PAP has been reported to be a potent inhibitor of sulfotransferases andas such has the potential to inhibit the same reaction which is beingassayed and in which it is produced. This aspect of sulfotransferasereactions makes development of an assay more complicated. However,because embodiments of the invention promptly degrade PAP to 5′-AMPusing gPAPP, this potential inhibition does not occur. As such, inaddition to providing a way to detect and quantify sulfotransferasereactions, embodiments of the invention avoid the inhibition ofsulfotransferase reactions which can be caused by the production of PAP.

Embodiments of the invention include assays, kits and methods fordetecting and measuring sulfotransferase activity. In some embodiments,the assay includes one or more of the following components: PAPS; gPAPP;a buffer; and a control sulfotransferase. The assay may optionallyinclude free phosphate detection reagents, such as a first reagentcomprising molybdate and a second reagent comprising malachite green. Insome embodiments, the assay may also include PAP.

In some embodiments, the kit may include gPAPP and a free phosphatedetection assay. In other embodiments, the kit may include PAPS andgPAPP. In still other embodiments, the kit may include gPAPP, a freephosphate detection assay, and PAPS. The kit may further include anassay buffer, PAP and/or a phosphate standard. The phosphate detectionassay may be a Malachite Green detection assay. The PAPS and/or PAP maybe supplied in the assay buffer. The gPAPP may also be supplied in theassay buffer.

In one embodiment, the sulfotransferase assay kit includes an assaybuffer, gPAPP, PAPS, Malachite Green Reagent A, Malachite Green ReagentB, and a phosphate standard, such as KH₂PO₄. The kit may further includea sulfotransferase to be used as a positive control for the variouscomponents of the kit. The gPAPP, PAPS and/or the controlsulfotransferase may be provided in the assay buffer. In someembodiments, the kit further includes a 5′-nucleotidase.

A sulfotransferase may be provided as a control in the kit, or a controlsulfotransferase may be supplied by the user of the kit. The controlsulfotransferase serves to assure proper functioning of the assay. Theassay may be performed using the control sulfotransferase and theresults may be compared to known expected results for thesulfotransferase. If the results are within the expected range, theassay can be considered to be functioning properly. In this way, whenthe assay is performed using a sulfotransferase of interest, the resultsmay be considered reliable. A control sulfotransferase provided in a kitis preferably stable over time and has a known activity, and the dataregarding the control sulfotransferase activity and expected results maybe provided with the kit. Examples of sulfotransferases which may beprovided in kits to serve as controls include any stablesulfotransferase having a high specific activity and an availablesubstrate, such as SULT1C4 and CHST3.

The use of the kit may include first creating a free phosphate standardcurve. For colorimetric assays, the phosphate standard curve may becreated using serial dilution of a phosphate standard in the assaybuffer, and followed by measuring the absorbance using a phosphatedetection reagents. For example, each dilution may be combined withMalachite Green Reagent A and then with Malachite Green Reagent B asdescribed and the absorbance may be read at 620 nm. The resultingmeasurements may be used to create a phosphate standard curve which maybe used to calculate the phosphate conversion factor, the amount ofphosphate corresponding to a unit of absorbance.

In order to correlate assay results to levels of free phosphate, andthereby to sulfotransferase activity, a phosphate standard curve may beproduced. In embodiments in which a Malachite Green assay is used tomeasure free phosphate, and in embodiments using other free phosphatedetection methods as well, the phosphate standard curve may be madeusing serial dilutions, such as 2-fold serial dilutions, of a phosphatesolution such as the phosphate standard, in the assay buffer. Forexample, the serial dilutions may be as in Table 1, below.

TABLE 1 Well Phosphate concentration (μm) Phosphate input (nmole) 1 505,000 2 25 2,500 3 12.5 1,250 4 6.25 625 5 3.13 313 6 1.56 156 7 0.78 788 0 0

When the Malachite Green assay is used, for example, the serialphosphate dilutions may be added to a clear 96-well plate and may beperformed in triplicate. The Malachite Green Reagent A is first added toeach well, followed by the Malachite Green Reagent B. After 20 minutes,the optical density (OD) is read at 620 nm for each well using amicroplate reader or spectrophotometer. The average OD for each dilutionmay be obtained. The phosphate input may be plotted against the OD, orthe average of the OD for each dilution, to create a standard curve,such as by using linear regression or a computer generated fourparameter logistic (4-PL) curve fit. A similar curve may be obtainedusing other free phosphate detection methods or other phosphate sources.The slope of the linear regression line may be used as the conversionfactor, i.e. the amount of phosphate corresponds to an absorbance unit.This conversion factor may then be used to calculate the amount of freephosphate from the measured absorbance for each reaction.

In some embodiments, a single buffer is used which is the assay buffer.The assay buffer should allow the sulfotransferase, and preferably alsogPAPP and the 5′-nucleotidase, if used, to function normally. In someembodiments, the assay buffer may contain about 10 mM MgCl₂ and may havea pH of about 7.0 to about 8.0. In some embodiments, the assay buffermay comprise 25 mM Tris and 10 mM MgCl₂ at pH 7.5. In other embodiments,two or more buffers may be used. The first buffer may be an assay bufferand the second buffer may be a phosphatase buffer. For example, thephosphatase may be Mg²⁺ dependent, and therefore the phosphatase buffershould include Mg²⁺, while the assay buffer might not have thiscomponent.

In some embodiments, the buffer and other reagents have divalent cationssuch as calcium, magnesium and manganese. In some embodiments, gPAPP(and the 5′-nucleotidase, if used) may be active in the assay bufferused with or provided with the assay. In such embodiments, gPAPP may becombined with the PAPS, the substrate, and the sulfotransferase, in thesame buffer. In other embodiments, gPAPP is not active in the assaybuffer used with or provided with the assay. In such embodiments, PAPS,the substrate, and the sulfotransferase may first be combined in a firstbuffer which is the assay buffer and allowed to react. A second bufferwhich is the gPAPP buffer may then be added after the completion of thefirst reaction. The gPAPP may be added with the gPAPP buffer or may beadded after the addition of a sufficient amount of the gPAPP buffer. ThegPAPP buffer may be stronger than the sulfotransferase assay buffer,such that the conditions provided by the gPAPP buffer will overwhelmthose provided by the assay buffer, with the resulting mixture beingmore similar to the gPAPP buffer and therefore being favorable for gPAPPactivity.

Embodiments of the invention provide convenient ways to detect andquantify sulfotransferase activity. The assays described herein do notinvolve radioisotope usage and do not require chemical separation, likeprevious methods. Furthermore, the assays described herein can beperformed using multi-well high throughput techniques.

Embodiments of the invention further include methods of detecting and/orquantifying the activity of a sulfotransferase. In some embodiments, themethod includes performing a first reaction including combining asulfotransferase, which may be considered a test sulfotransferase, witha substrate of the sulfotransferase and PAPS in a reaction buffer. Themethod further includes combining the reactants with gPAPP. In someembodiments, the gPAPP may be combined simultaneously or approximatelysimultaneously with the other reactant. In other embodiments, thesulfotransferase, substrate and PAPS are combined first and allowed toreact, and then the gPAPP is added to the reaction after the reactionhas progressed for a certain amount of time, such as about 20 minutes.

In some embodiments, the method includes performing a first reactionincluding combining a sulfotransferase which is a test sulfotransferasewith a substrate of the sulfotransferases and PAPS in a reaction buffer.The method further includes combining the reactants with gPAPP and a5′-nucleotidase. In some embodiments, the gPAPP and the 5′-nucleotidasemay be combined simultaneously or approximately simultaneously with theother reactants. In other embodiments, the gPAPP and the 5′nucleotidaseare combined with the other reactants after the sulfotransferasereaction has progressed for a certain amount of time, such as about 20minutes. In still other embodiments, the gPAPP is added to the reactionfirst either simultaneously or after allowing the sulfotransferasereaction to progress and then the 5′-nucleotidase is added afterallowing time for the gPAPP to react.

The next step in any of the embodiment is measuring the level of freephosphate. In some embodiments, the reaction is tested using a freephosphate detector such as the Malachite Green assay as described above.In such embodiments, malachite reagent A is added first, followed by theaddition of malachite reagent B.

In any of the methods described above, measuring the level of freephosphate may include measuring OD and comparing the measured OD to afree phosphate standard curve or applying a conversion factor which maybe determined using a free phosphate standard curve to the measured OD,to convert the measured OD to a free phosphate level. In suchembodiments, the method may further include preparing the free phosphatestandard curve.

In any of the methods described above, the method may further includeperforming a second reaction which is a control reaction. In someembodiments, performing the control reaction includes combining each ofthe reactants as in the first reaction (the test reaction) in the samemanner and under the same conditions as the test reaction but in theabsence of the sulfotransferase. For example, performing the secondreaction may include combining the substrate of the sulfotransferasewith PAPS, and either concurrently or subsequently adding gPAPP,depending upon how the test reaction was performed. If used in the testreaction, the 5′-nucleotidase would also be combined with the reactants,in the same manner as in the test reaction. The method further includesmeasuring the level of free phosphate in the second reaction, and thisstep would be performed in the same manner as in the test reaction.Finally, the method may include subtracting the measured free phosphateof the second reaction from the measured free phosphate of the firstreaction. In this way, the results can be adjusted to compensate for anyfree phosphate, PAP and 5′-AMP that may be present in the reagents andare not caused by the sulfotransferase reaction. For example, someamount of PAPS may spontaneously degrade to PAP even in the absence of asulfotransferase, making the PAP available to the gPAPP and resulting insome amount of free phosphate. By adjusting (reducing) the level of thefree phosphate measured in the sulfotransferase test reaction by theamount of free phosphate present in the control, a more accuratemeasurement of sulfotransferase activity can be obtained which does notdepend upon the stability of PAPS.

In some embodiments, the assay is used to evaluate the effect of a testagent on the sulfotransferase. In such embodiments, thesulfotransferase, substrate, PAPS and test agent are combined in a testreaction, and gPAPP may be added simultaneously or subsequently. In someembodiments, a 5′nucleotide may also be added to the reaction. Next thephosphate level is measured as in the other methods. The phosphate level(or the activity of the sulfotransferase which may be determined usingthe phosphate level) is compared to the phosphate level (orsulfotransferase activity) for a separate reaction (a control reaction)including the same reactants but without the test agent. The controlreaction may either be performed or its values (phosphate level and/orsulfotransferase activity) may already be known and used for comparisonto the test reaction.

EXAMPLES

In the following examples, the gPAPP used was recombinant mouse gPAPPexpressed in CHO cells as an N-terminal His-tagged recombinant proteincontaining amino acids from E51 to K356, created by R & D Systems. Thesequence was based on accession number NP 808398 from the NationalCenter for Biotechnology Information. The chondroitin sulfate (CS) andp-nitrophenol (pNP) were obtained from Sigma Aldrich. The PAPS wasprepared by R & D Systems using recombinant S. cerevisiae ATP sulurylaseand P. chrysogenum APS kinase. The APS kinase was also prepared by R & DSystems. Recombinant human SULT1A1, CHST3, CD73, TNAP and MalachiteGreen Phosphate Detection Kit were from R & D Systems. Proteinconcentrations were quantified using the Bradford assay as described byBradford, M. M., A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem. 1976. Vol 72, Page 248-254, the relevant portionsof which are hereby incorporated by reference. Phosphate levels weredetermined using the Malachite Green Phosphate Detection Kit availablefrom R & D Systems, according to the package instructions. All exampleswere performed in a buffer of 25 mM Tris at pH 7.5.

Example 1

First, the activity of gPAPP in the presence of Mg²⁺ was tested bycombining 0.025 μg gPAPP, 25 nmol PAP and increasing amounts of Mg²⁺ in50 μL of assay buffer of 25 mM Tris at pH 7.5. The results are shown inFIG. 4. As can be seen, gPAPP was found to be active in the presence ofMg²⁺, and a maximum activity was achieved when the Mg²⁺ concentrationwas greater than 10 mM. The magnesium dependence of gPAPP is consistentwith its localization in the Golgi apparatus and the presence ofmagnesium transporters in the Golgi apparatus. In a second experiment,the activity of gPAPP was evaluated at various pH levels. The activityof gPAPP in the presence of 10 mM Mg²⁺ was tested by combining 0.025 μggPAPP, 25 nmol PAP in 50 μL of buffers at different pH levels. It wasfound that gPAPP was active at a pH from 4.0 to 9.0, with optimalactivity around pH 7.5.

Example 2

The specificity of gPAPP was first tested on 3′-AMP, 5′-AMP and PAP inthe absence or presence of CD73 by combining 10 nmol of either 3′-AMP,5′-AMP or PAP with 1 μg gPAPP and/or 0.1 μg CD73 in 50 μL assay buffercontaining 10 mM Mg²⁺ at pH 7.5. In a parallel experiment, the sameamount of nucleotide was combined with tissue-non-specific alkalinephosphatase (TNAP) at pH 9.0. The ratio between the phosphate releasedby gPAPP and/or CD73 (Pi) and the phosphate released by TNAP (Pi_(TNAP))was plotted versus the nucleotide and phosphatases in FIG. 5. It can beseen that gPAPP alone showed activity on PAP but not 3′-AMP or 5′-AMP.When gPAPP and CD73 were used together, they released twice as muchphosphate from PAP than gPAPP alone. These results confirmed that gPAPPis a PAP-specific 3′-nucleotidase and CD73 is a 5′-nucleotidase.

Example 3

The specificity of gPAPP was tested on PAP and PAPS. These results areshown in FIG. 6. In this example, 50 nmol of either PAP or PAPS wastreated with different amounts of gPAPP in 50 μL assay buffer containing10 mM Mg²⁺ at pH 7.5 for 20 minutes. The released phosphate was plottedversus the amount of gPAPP. While the phosphate released from PAP bygPAPP increased with the amount of the enzyme, the phosphate releasedfrom PAPS by gPAPP showed no change. The results of examples 1-3demonstrate that gPAPP can be used as a coupling phophatase insulfotransferase assays to release the 3′-phosphate from PAP, and thatCD73 can be used as a secondary coupling phosphatase to further releasethe 5′-phosphate from 5′-AMP produced in a gPAPP-coupledsulfotransferase reaction.

Example 4

In this example, the coupling capacity of gPAPP was determined. Thecoupling capacity of a coupling enzyme is the amount of product that canbe completely converted into signal by one μg of the enzyme underspecific conditions. In this example, the coupling capacity is theamount of PAP (product) that can be converted by gPAPP into signal(phosphate). The coupling capacity of gPAPP was determined by combining1 μg gPAPP with increasing amounts of PAP, in the presence or absence of0.5 mM PAPS in 50 μL assay buffer containing 10 mM Mg²⁺ at pH 7.5 foreither 20 or 40 minutes. The released phosphate was then plotted versusPAP input as shown in FIG. 7. It was found that 1 μg PAPP was able tocomplete the hydrolysis of 8 nmol and 15 nmol of PAP in 20 and 40minutes, respectively. The coupling capacity for gPAPP was therefore 8nmol in a 20 minute reaction and 15 nmol in a 40 minute reaction. Thepresence of PAPS did cause about a 20% inhibition of the hydrolysis whenthe amount of PAP was greater than the coupling capacity, but PAPS didnot change the coupling capacity significantly, as the PAP-hydrolysiscurve in the presence of PAPS almost overlaps with the PAP-hydrolysiscurve in the absence of PAPS, when PAP concentration is below thecoupling capacity.

Example 5

In this example, p-nitrophonol (pNP) was used as a substrate. Thesulfotransferase SULT1A1 is known to have a high affinity for pNP, witha half maximal velocity (Km) reported below 1 μM. Measuring the Km ofSULT1A1 for pNP therefore requires a highly sensitive test. Because ofthis, CD73 was used in the reaction in addition to gPAPP, to increasethe assay sensitivity.

A series of reactions was performed by combining various amounts ofSULT1A1 with 0.25 mM PAPS and 0.25 mM pNP and with either 1 μg gPAPPalone, or both 1 μg gPAPP and 0.1 μg CD73 in 50 μL of Tris buffercontaining 10 mM of MgCl₂ at pH 7.5. The rate of phosphate release wasthen plotted versus the enzyme input as shown in FIG. 8. While the rateof phosphate release when coupled to gPAPP alone was 38.1 pmol/min/μg,the rate increased to approximately two fold to 83.7 pmol/min/μg whencoupled to both gPAPP and CD73. Since gPAPP and CD73 together releasetwo equivalents of phosphate from each PAP, the specific activity ofSULT1A1 was averaged to 40.0±1.9 pmol/min/gg. This specific activity isconsistent with the known results obtained using radioisotope assays.

Example 6

A series of reactions were performed by combining increasingconcentrations of pNP with 0.1 μg SULT1A1, 2 μg PAPP and 0.1 μg CD73 in200 μL of an assay buffer containing 0.25 mM PAPS, 10 mM MgCl₂ at pH7.5. The free phosphate was measured and the reaction velocity wasplotted against pNP concentration as shown in FIG. 9. At pNPconcentrations above 4 mM, substrate inhibition was present and theconcentration for half maximum velocity was projected to be <1.0 μM ofpNP. These values are consistent with reported values for SULT1A1determined using radioisotope assays in the literature.

Example 7

Carbohydrate sulfotransferase 3 (CHST3) is a sulfotransferase which isknown to catalyze the sulfation of N-acetylgalactosamine at the 6-Oposition. In a first series of reactions, 1 μg CHST3 was combined with 1μg gPAPP and increasing concentrations of CS in 50 μl assay buffercontaining 0.8 mM PAPS, 10 mM MgCl₂ at pH 7.5. In a second series ofreactions, 0.4 μg CHST3 was combined with 1 μg gPAPP and increasingconcentrations of PAPS in 50 μl assay buffer containing 0.8 mM PAPS, 10mg/ml CS, 10 mM MgCl₂ at pH 7.5. Under the conditions of the firstseries of reactions, it was determined that the concentration of CS thatlead to a half-maximal velocity (Km for CS) was about 2 mg/ml, as shownin FIG. 10A. Under the conditions of the second series of reactions, thePAPS concentration for half-maximal velocity (Km for PAPS) was below 0.1mM, as shown in FIG. 10B. It can be seen that substrate inhibitionoccurred at PAPS levels above 0.5 mM in FIG. 10B, while no substrateinhibition is seen in FIG. 10A.

Example 8

First, the optimal concentration for CS was visually determined to be >5mg/ml and the optimal concentration for PAPS was visually determined tobe about 0.5 mM using FIGS. 10A and 10B by finding the substrateconcentration corresponding to the highest point in the curves. Anenzyme dose curve was then created for CHST3 at the optimalconcentrations for both the donor and acceptor substrates in FIG. 11. Tocreate the dose curve, 1 μg gPAPP was combined with increasing amountsof CHST3 in the presence of 10 mg/ml of CS and 0.5 mM of PAPS in 50 μlassay buffer containing 10 mM MgCl₂ at pH 7.5. The specific activity wasdetermined to be 1110 pmol/min/gg, as shown in FIG. 11, which isconsistent with data previously obtained using a gel electrophoresisbased radioisotope assay.

In the foregoing detailed description, the invention has been describedwith reference to specific embodiments. However, it may be appreciatedthat various modifications and changes can be made without departingfrom the scope of the invention as set forth.

Embodiments of the invention includes various aspects, some of which aredescribed in the numbered paragraphs below.

1. An assay for detecting activity of a test sulfotransferasecomprising: gPAPP and a free phosphate detector. The free phosphatedetector may be a colorimetric assay, such as a free phosphate detectorincluding a first reagent and a second reagent, wherein the firstreagent comprises ammonium molybdate and the second reagent comprisesmalachite green oxalate. The assay may further include a source of freephosphate for use as a phosphate standard and/or PAPS and/or PAP. Theassay may further include a buffer, which may include magnesium.

2. An assay for detecting activity of a test sulfotransferasecomprising: gPAPP and a control sulfotransferase. The controlsulfotransferase may be SULT1C4 or CHST3, for example. The assay mayfurther include a source of free phosphate for use as a phosphatestandard and/or PAPS and/or PAP. The assay may further include a buffer,which may include magnesium.

3. An assay for detecting activity of a test sulfotransferasecomprising: gPAPP; a control sulfotransferase; and a free phosphatedetector. The free phosphate detector may be a colorimetric assay, suchas a free phosphate detector including a first reagent and a secondreagent, wherein the first reagent comprises ammonium molybdate and thesecond reagent comprises malachite green oxalate. The controlsulfotransferase may be SULT1C4 or CHST3, for example. The assay mayfurther include a source of free phosphate for use as a phosphatestandard and/or PAPS and/or PAP. The assay may further include a buffer,which may include magnesium.

4. An assay for detecting activity of a test sulfotransferasecomprising: gPAPP; a 5′ nucleotidase; and a free phosphate detector. Theassay may further include PAPS and/or PAP.

An assay for detecting activity of a test sulfotransferase comprising:gPAPP; a 5′ nucleotidase; and a control sulfotransferase. The assay mayfurther include PAPS and/or PAP.

An assay for detecting activity of a test sulfotransferase comprising:gPAPP; a 5′ nucleotidase; a control sulfotransferase, and a freephosphate detector. The assay may further include PAPS and/or PAP.

5. An assay for detecting activity of a test sulfotransferasecomprising: gPAPP, a control sulfotransferase, a free phosphatedetector, PAPS and a buffer. The assay may further include PAP and/or a5′ nucleotidase.

6. A method of detecting sulfotransferase activity comprising conductinga first reaction comprising: combining a sulfotransferase, gPAPP, asubstrate of the sulfotransferase, and PAPS under conditions to producePAP; and measuring free phosphate. The first reaction can furtherinclude a buffer, which may include magnesium. Measuring free phosphateincludes applying a colorimetric assay to the first reaction andmeasuring optical density.

7. A method of detecting sulfotransferase activity comprising conductinga first reaction comprising: combining a sulfotransferase, gPAPP, asubstrate of the sulfotransferase, and PAPS under conditions to producePAP; measuring free phosphate; and calculating sulfotransferase activityusing the measured amount of free phosphate. The first reaction canfurther include a buffer, which may include magnesium. Measuring freephosphate includes applying a colorimetric assay to the first reactionand measuring optical density.

8. A method of detecting sulfotransferase activity comprising conductinga first reaction and a second reaction. The first reaction comprisescombining a sulfotransferase, gPAPP, a substrate of thesulfotransferase, and PAPS under conditions to produce PAP; andmeasuring free phosphate. Measuring free phosphate includes applying acolorimetric assay to the first reaction and measuring optical density.The first reaction can further include a buffer, which may includemagnesium. The second reaction comprises combining the substrate of thesulfotransferase and the PAPS with the phosphatase in the absence of thesulfotransferase and measuring free phosphate, such as under the sameconditions as the first reaction (except for the absence ofsulfotransferase), wherein the second reaction provides a backgroundcontrol for the first reaction. The second reaction can include the samebuffer as the first reaction. The method can further include reducingthe measured phosphate of the first reaction by the measured phosphateof the second reaction to calculate the amount of phosphate correlatedto the sulfotransferase reaction.

9. A method of detecting sulfotransferase activity comprising conductinga first reaction comprising: combining a sulfotransferase, gPAPP,5′-nucleotidase, a substrate of the sulfotransferase, and PAPS underconditions to produce PAP; and measuring free phosphate. Measuring freephosphate includes applying a colorimetric assay to the first reactionand measuring optical density.

10. A method of testing an agent for effect upon a sulfotransferasecomprising: combining a sulfotransferase, gPAPP, a substrate of thesulfotransferase, the agent and PAPS under conditions to produce PAP;measuring free phosphate; and calculating sulfotransferase activityusing the measured amount of free phosphate. The reaction can furtherinclude a buffer, which may include magnesium. Measuring free phosphateincludes applying a colorimetric assay to the first reaction andmeasuring optical density. The method can further include performing thesame reaction in the absence of the agent, and comparing the results ofthe two reactions to determine the effect of the agent uponsulfotransferase activity.

1. A method of detecting sulfotransferase activity comprising conductinga first reaction comprising: combining a sulfotransferase,Golgi-resident PAP-phosphatase (gPAPP), a 5′-nucleotidase, a substrateof the sulfotransferase, and 3′-phosphoadenosine-5′-phosphosulfate(PAPS) under conditions to produce free phosphate; measuring freephosphate; and comparing the measured free phosphate to a free phosphatestandard curve or equation or to a free phosphate level obtained in aseparate reaction.
 2. The method of claim 1 further comprisingcalculating sulfotransferase activity using the measured amount of freephosphate.
 3. The method of claim 1 wherein the step of comparingcomprises comparing the measured free phosphate to a free phosphatelevel obtained in a separate reaction, wherein the separate reactioncomprises a background control reaction wherein the (gPAPP), the5′-nucleotidase, the substrate of the sulfotransferase, and the (PAPS)were combined without the sulfotransferase.
 4. The method of claim 3further comprising reducing the measured phosphate of the reaction bythe measured phosphate of the background control reaction to calculatethe amount of phosphate correlated to the sulfotransferase reaction. 5.The method of claim 4 further comprising calculating sulfotransferaseactivity using the calculated the amount of phosphate correlated to thesulfotransferase reaction.
 6. The method of claim 1 wherein the step ofcomparing comprises comparing the measured free phosphate to a freephosphate level obtained in a separate reaction, wherein the separatereaction comprises comprises a reaction wherein the sulfotransferase,the gPAPP, the 5′-nucleotidase, the substrate of the sulfotransferase,and the PAPS were combined and wherein one or more conditions of theseparate reaction were different from those of the reaction.
 7. Themethod of claim 1 wherein measuring the free phosphate comprisesmeasuring optical density.
 8. The method of claim 1 wherein measuringfree phosphate comprises applying a colorimetric free phosphatedetection assay to the first reaction.
 9. The method of claim 1 whereinmeasuring free phosphate comprises adding a first reagent to thereaction comprising ammonium molybdate and then adding a second reagentto the reaction comprising malachite green oxalate.
 10. The method ofclaim 1 wherein the step of comparing comprises comparing the measuredfree phosphate to a free phosphate standard curve.
 11. The method ofclaim 10 further comprising calculating sulfotransferase activity usingthe measured amount of free phosphate.